The present application is a 371 U.S. national phase application of PCT/EP99/04342, filed Jun. 23, 1999.
The present invention is based on the finding that the envelope proteins of HCV induce a beneficial immune response in chimpanzees which are chronically infected with a heterologous subtype 1a or subtype 1b HCV strain. More specifically, the present invention relates to the finding that envelope proteins are highly immunogenic and result in the stimulation of both the cellular and humoral immune response. Moreover, the present invention relates to the finding that blocking of cysteines by alkylation results in even more immunogenic proteins. In addition, the envelope proteins of the present invention can be incorporated in particles which display a high imnmunogenecity and immunoreactivity. It was further demonstrated that such particles may incorporate other proteins.
Hepatitis C virus (HCV) infection is a major health problem in both developed and developing countries. It is estimated that about 1 to 5% of the world population is affected by the virus. HCV infection appears to be the most important cause of transfusion-associated hepatitis and frequently progresses to chronic liver damage. Moreover, there is evidence implicating HCV in induction of hepatocellular carcinoma. Consequently, the demand for reliable diagnostic methods and effective therapeutic agents is high. Also sensitive and specific screening methods of HCV-contaminated blood-products and improved methods to culture HCV are needed.
HCV is a positive stranded RNA virus of approximately 9,600 bases which encode at least three structural and six non-structural proteins. Based on sequence homology, the structural proteins have been functionally assigned as one single core protein and two envelope proteins: E1 and E2. The E1 protein consists of 192 amino acids and contains 5 to 6 N-glycosylation sites, depending on the HCV genotype. The E2 protein consists of 363 to 370 amino acids and contains 9-11 N-glycosylation sites, depending on the HCV genotype (for reviews see: Major and Feinstone, 1997; Maertens and Stuyver, 1997). The E1 protein contains various variable domains (Maertens and Stuyver, 1997), while the E2 protein contains three hypervariable domains, of which the major domain is located at the N-terminus of the protein (Maertens and Stuyver, 1997). The latter envelope proteins have been produced by recombinant techniques in Escherichia coli, insect cells, yeast cells and mammalian cells. The usage of an expression system in higher eukaryotes and especially in mammalian cell culture leads to envelope proteins that are effectively recognized by antibodies in patient samples (Maertens et al., 1994).
It has been suggested that the E1 envelope protein needs the E2 envelope protein to reach a proper folding status (Deleersnyder et al., 1997). In addition, it has been suggested that E1 and E2 form heterodimers which may form the basic unit of the viral envelope (Yi et al., 1997). In WO 98/21338 to Liang et al. these presumptions have been used to construct HCV particles, which consist of E1 and E2, as well as Core and P7. In other words, the usage of E1 or E2 separately for immunization and other purposes is not suggested in the prior art. But, Houghton (1997) reported that repeated immunizations with recombinant gpE1E2 (4xc3x9725 xcexcg) of 3 chronically HCV-infected chimpanzees did not induce a significant immune response. The inventors of the present application reasoned that the induction of an anti-envelope immune response in patients with chronic hepatitis C would indeed be desirable and beneficial to the patient, since higher levels of such antibodies seem to correlate with good response to interferon therapy, and may therefore help the patient to clear the virus (PCT/EP 95/03031 to Maertens et al.). The inventors of the present invention further reasoned that, as the antibody levels against E1 in chronic HCV carriers are among the lowest of all HCV antibodies, it may be beneficial to raise those antibody levels, and possibly the cellular response, to induce control or even clearance of the infection by the host. Also higher levels of cellular immunity against E1 seem to correlate with good response towards interferon therapy (Leroux-Roels et al., 1996).
Besides the importance of anti-E1 immunity in relation to interferon therapy, other indications point-out that some other parts of the HCV genome may be important to induce a specific immune response which may allow control of the infection. Also T-cell reactivity against the C-terminal region of the core protein has been observed more frequently in patients responding to interferon therapy (Leroux-Roels et al, 1996). Potentially neutralizing antibodies against the NS4B protein were demonstrated in patients clearing HCV after liver transplant (Villa et al., 1998). Also within NS3 several T-cell epitopes have been mapped which seem to correlate with clearing of HCV during the acute phase (see: PCT/EP 94/03555 to Leroux-Roels et al.; Leroux-Roels et al., 1996; Rehermann et al., 1996 and 1997; Diepolder et al., 1995and 1997). Furthermore, antibodies to NS5A, like E1 antibodies, show higher levels at baseline before interferon-alpha therapy in long term responders (LTR) as compared to non-responders.
At present, therapeutic vaccination for HCV has not been successful. Also prophylactic vaccination has only been shown to be effective against a homologous strain of HCV (Choo et al., 1994). The present invention relates to the surprising finding that administration of an HCV envelope antigen can dramatically improve the state of chronic active hepatitis in an individual infected with a heterologous strain or isolate, both in a heterologous subtype 1a or heterologous subtype 1b infection. Indeed, chronically infected chimpanzees who were administered six doses of 50 xcexcg E1s (i.e. aa 192-326 of E1) surprisingly showed vigourous humoral and cellular immune responses, which had not been mounted over the entire period of chronic infection before the latter vaccination. Moreover, viral antigen became undetectable in the liver over a period of two to five months and remained undetectable for at least 5 months post-vaccination. Although HCV-RNA titers in the serum did not decrease, liver enzyme levels in the serum showed a clear tendency to normalize. Most importantly, liver histology improved dramatically in both vaccinees. The present invention further relates to the surprising finding that the E1 protein used for vaccination, which was expressed as a single HCV protein without its hydrophobic anchor, forms stable particles. It should also be noted that, to avoid induction of an immune response against non-relevant epitopes, the E1 protein used for vaccination was constructed as a consensus sequence of individual clones derived from a single serum sample from one chronic carrier. In addition, the present application relates to the finding that the induction of such anti-E1 responses may be increased by using antigens of a different genotype than the ones of the infection present in the host. Moreover, the present application relates to the finding that when cysteines of HCV envelope proteins are alkylated, for instance ,by means of N-(iodoethyl)-trifluoroacetamide, ethylenimine or active halogens, such as iodoacetamide, the oligomeric particles as described above display an even higher immunogenicity. Finally, the present invention relates also to the finding that mutation of cysteines of HCV envelope proteins to any other naturally occuring amino acid, preferentially to methionine, glutamic acid, glutamine or lysine, in the oligomeric particles as described above also results in higher immunogenicity, compared to the originals envelope proteins.
It is clear from the literature that there is an urgent need to develop reliable vaccines and effective therapeutic agents for HCV. Therefore, the present invention aims at providing an antigen preparation, which is able to induce specific humoral and cellular immunity to HCV envelope proteins, even (but not solely) in chronic HCV carriers. The same antigens can be used for diagnosis of the immune response.
More specifically, the present invention aims at providing an antigen preparation as defined above, which consists of stable particles of single envelope proteins of HCV. It should be clear that, at present, such particles or a method to prepare such particles, are not known in the art. Moreover, there is no indication in the art that any antigen preparation, including such stable particles or such purified single HCV envelope proteins, could successfully be used as (heterologous) prophylactic or therapeutic vaccine against HCV. The present invention thus also aims at providing a method to produce stable particles, which can be successfully used as a prophylactic or therapeutic agent against HCV, in addition to provide DNA vaccines encoding HCV antigens. More specifically, the present invention aims at providing a method to produce the latter particles based on detergent-assisted particle formation (see further). Furthermore, the present invention aims at providing methods to prepare particles consisting of antigens obtained from different HCV genotypes.
Moreover, the present invention aims at providing an antigen which is a consensus sequence from individual clones, which may allow a more correct folding of the proteins. This in order to avoid stimulation of immunity against non-relevant epitopes.
Furthermore, the present invention aims at providing an antigen formulation, in particular for therapeutic vaccination, based on the genotype of HCV by which the chronic carrier is infected. In this regard, the present invention aims at providing an envelope protein of either a different or a homologous genotype or subtype compared to the genotype or subtype of the chronic carrier.
A further aim of the invention is to provide a method for treating or therapeutically vaccinating chronically infected patients using the above-indicated antigens or DNA vaccines, possibly in combination with other compounds. The present invention also aims to provide a method to prophylactically vaccinate humans against HCV.
Another aim of the invention is to provide oligomeric particles which have a superior immunogenicity, due to the mutation of at least one cysteine residue of HCV envelope protein into a natural occuring amino acid, preferentially methionine, glutamic acid, glutamine or lysine. Alternatively, alkylation of at least one cysteine residue of HCV envelope protein may be performed. In particular, the latter protein can be alkylated by means of ethylenimine, N-(iodoethyl)trifluoroacetamide or active halogens. In this regard, the instant invention aims to provide the additional use of oligomeric particles as vehicles for presenting non-HCV epitopes efficiently.
It is a further aim of the present invention to provide a method to treat patients, acutely or chronically infected, with an anti-envelope antibody, such as anti-E1 antibody, e.g. anti-E1 V2 region antibody, either alone or in combination with other treatments.
Another aim of the invention is to provide a T cell stimulating antigen such as Core, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, or NS5B along with the envelope proteins of the invention.
All the aims of the present invention are considered to have been met by the embodiments as set out below.